title: Limited proteolysis and site-directed mutagenesis reveal the origin of microheterogeneity in Rhodotorula gracilis D-amino acid oxidase.
abstract: When analysed by isoelectric focusing, D-amino acid oxidase from the yeast Rhodotorula gracilis normally consists of three molecular isoforms (pI 7.8, 7.4 and 7.2, respectively) all with the same N-terminal sequence. However, only a single band of pI 7.8 is detected with the recombinant wild-type protein expressed in E. coli. To determine whether the molecular basis of this heterogeneity is due to proteolysed forms of the protein, we treated R. gracilis D-amino acid oxidase with various proteases. Limited proteolysis by chymotrypsin and thermolysin produced truncated and nicked monomeric holoenzymes containing two polypeptides of approximately 34 kDa (Met1-Leu312) and one of approximately 5 kDa (Ala319-Arg364 with chymotrypsin or Ala319-Ala362 with thermolysin). On the other hand, treatment with endoproteinase Glu-C gave a dimeric holoenzyme lacking the C-terminal SKL tripeptide. This cleavage of Glu365-Ser366 peptide bond caused the disappearance of the three isoelectric bands and a single homogeneous band (pI 7.2) appeared. To study this protein form, we used site-directed mutagenesis to produce a mutant form of R. gracilis D-amino acid oxidase lacking the SKL C-terminal tripeptide (which is the targeting sequence PTS1 for peroxisomal proteins). As expected, the SKL-deleted mutant gave a single band (pI 7.2) in isoelectric focusing. The three-band pattern of native yeast enzyme was generated by in vitro experiments using an equimolar mixture of the wild-type (pI 7.8) and the SKL-deleted recombinant (pI 7.2) DAAOs. The microheterogeneity of yeast DAAO thus stems from the association of two polypeptide chains differing in the C-terminal tripeptide, giving three different holoenzyme dimers.
authors.affiliation: Department of Structural and Functional Biology, University of Milano, via Ravasi 2, 21100 Varese, Italy.
authors.forename: S
authors.initials: S
authors.lastname: Campaner
authors.forename: L
authors.initials: L
authors.lastname: Pollegioni
authors.forename: B D
authors.initials: BD
authors.lastname: Ross
authors.forename: M S
authors.initials: MS
authors.lastname: Pilone
mesh_terms: D002918:Chymotrypsin / Q000378:metabolism; D003605:D-Amino-Acid Oxidase / Q000737:chemistry* / Q000235:genetics; D019281:Dimerization; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D012248:Rhodotorula / Q000201:enzymology*; D013820:Thermolysin / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D007527:Isoenzymes; D003605:D-Amino-Acid Oxidase; D002918:Chymotrypsin; D013820:Thermolysin